Guedelhoefer Otto C, Sánchez Alvarado Alejandro
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Utah, USA.
J Vis Exp. 2012 Aug 6(66):4015. doi: 10.3791/4015.
The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed. Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration. Both tissue transplantation and partial irradiation have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.
涡虫，一种淡水扁虫，已被证明是剖析后生动物再生和干细胞生物学的强大系统。涡虫缺失或受损组织的再生是由称为新成细胞的成体干细胞实现的。尽管这些干细胞已被明确证明具有多能性，且唯一能够重建整个动物个体，但干细胞群体内部的异质性及其细胞行为的动态变化在很大程度上仍未得到解决。由于干细胞在涡虫整个身体结构中的数量众多且分布广泛，因此需要先进的方法来操纵干细胞亚群，以便在体内进行分子和功能研究。组织移植和局部辐照是两种可以分离涡虫干细胞亚群以进行进一步研究的方法。每种技术都有其独特的优势。组织移植能够将本质上基因不同或先前经过药物处理的干细胞引入未接触过的宿主。或者，局部辐照能够在宿主体内分离出与不含干细胞的组织相邻的干细胞，而无需造成伤口或破坏组织完整性。使用这两种方法，可以研究控制干细胞功能（如增殖、分化和迁移）的细胞自主和非自主因素。组织移植和局部辐照在历史上都被用于确定许多至今仍在研究的关于涡虫再生的问题。然而，由于先前方法繁琐且不一致的性质，这些技术的使用一直不足。这里介绍的方案在减少可重复产生大量移植或局部辐照动物所需的时间和精力方面向前迈出了一大步，其效率接近100%。我们涵盖了大型动物的培养、固定、局部辐照准备、组织移植以及动物恢复的优化。此外，这里描述的工作展示了局部辐照方法首次应用于研究最广泛的涡虫——地中海涡虫。此外，涡虫中高效的组织移植为在再殖实验中对未接触过的或经过处理的干细胞亚群进行功能测试打开了大门，这长期以来一直是检测哺乳动物成体干细胞潜能的金标准方法。广泛采用这些技术无疑将有助于更好地理解成体干细胞在组织稳态和再生过程中的细胞行为。
